This proposal is to supplement grant K08DK02387, The molecular basis of adrenarche: dissection of P450c17 activities. The work during years 01 and 02 of the parent award have yielded a completed computer model of human P450c17 (17alpha- hydroxylase/17, 20-lyase) that has identified important residues for the interaction of P450c17 with redox partner proteins. Analyses of this model and of sequence alignments reveal what we believe to be the residues in the active site responsible for conveying either 17alpha-hydroxylase or 21-hydroxylase activity onto the P450 structural template. We will test this prediction by constructing P450c17-P450c21 hybrid proteins. We have also shown that cytochrome b5 (b5) selectively augments the 17,20- lyase activity of human P450c17 by acting as an allosteric facilitator of the P450c17 P450-OR complex rather that serving as an alternate electron donor. Based on our analysis of the structure of bovine and rat cytochromes b5, we propose to study truncated and mutant forms of cytochrome b5 to identify the minimal functional component of human b5 that stimulates the 17,20-lyase activity of P450c17. These studies will define functional regions of P450c17 and of b5 that differentially participate in the regulation of the 17alpha-hydroxylase and 17,20-lyase activities, which will enable novel approaches to pharmacological manipulation of these activities. While recognizing the central role of P450c17 in the biosynthesis of sex steriods, it is also true that the rise in DHEA-S production that defines adrenarche cannot occur unless pregnenolone, generated by the action of P450scc on cholesterol in the adrenal mitochondria, can escape the action of 3beta-hydroxysteroid dehydrogenase/delta5-4-isomerase type II (3betaHSDII), located throughout the adrenal cell, and bind to microsomal P450c17 for two successive turnovers. We hypothesize that a post- translational modification of 3betaHSDII can regulate the subcellular localization of 3betaHSDII and that subcellular localization rather than quantity of enzyme alone controls the flux of steroid precursors down the delta5 and delta4 pathways. We therefore propose to determine if 3betaHSDII is phosphorylated or acylated. We will use our yeast expression system to study the dynamics and regulation of 3betaHSDII localization in a model adrenal cell system. In completing this work, the PI will test a hypothesis of a novel regulatory mechanism for determining flux along specific steroidogenic pathways and commence an area of investigation that complements his previous work under this proposal and defines his own line of independent investigation.